Today cryo electron microscopy can be used to determine the 3 dimensional structures of macromolecules at near atomic resolution. EM can be used to get diffraction from 2D crystals or images in 2D of macromolecules that can be reconstructed into 3D by using 2D images from different angles. The first method is usually referred to as EM crystallography and the latter can be divided into electron tomography and single-particle electron microscopy. The electron tomography approach requires many pictures of the same particle tilted into different orientations. The single particle technique, collect 2D images of objects without tilting the specimen. The method exploits random orientations that particles assume in vitrified solvent to extract 3D information. A difference between the two methods is that when the specimen is tilted, 3D reconstructions of objects represent a single object in contrast to when using zero-tilt images the 3D reconstructed object represents an average of similar objects.
When the specimen is tilted, the relationship between the images and therefore the objects in the images are known by the tilt angle making the reconstruction of the object relatively straightforward. For zero-tilt techniques the relationship between the images and the objects in the images are unknown which leads to difficulties in relating the different objects to each other. A second complication for zero tilt techniques is that macromolecules are dynamic so that the objects in the images represent different conformations of the molecules. This adds another difficulty in the 3D reconstruction of macromolecules from zero tilt series data sets.
A major problem with the zero-tilt technique is to infer the orientations of the particles in the 2D images to enable a 3D structure to be determined. A number of techniques have been developed for an overview see (Angular Reconstitution in Three-Dimensional Electron Microscopy Historical and Theoretical Aspects, Scanning Microscopy Vol. 11 1997:195-210). None of the techniques that have been developed uses experimental information to establish the relationship between 2D images of random orientation. Structures without internal symmetry have been determined at resolutions of 5-9 Å, using zero tilt techniques.
Another major problem with the zero tilt techniques is that knowledge is lacking about how the macromolecules look in 3D before the molecular objects are oriented and reconstructed. As proteins are dynamic it is expected that the 2D molecular images represents different conformations of the molecule (sometimes referred to as conformers), and therefore there is a big risk that the 3D reconstructed objects will originate from 2D molecular images of different conformations, which will limit the resolution that can be achieved. This has been recognized by Gao et al, (Dynamics of EF-G interaction with the ribosome explored by classification of a heterogeneous cryo-EM dataset. J of Struct. Biol. 147, 2004, 283-290) where known differences have been used to make sub selections of molecular images that was used for reconstructions.
There is a need for a tool that enables the orientation of different conformations of a macromolecule so that the 3D reconstruction from 2D zero tilt images only contain images representing one single conformation.